Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides

ABSTRACT

The present invention relates to mutated and/or transformed microorganisms for the synthesis of various compounds. More specifically, the present invention discloses microorganisms mutated in the genes encoding for the regulators ArcA and IclR. The latter mutations result in a significant upregulation of the genes that are part of the colanic acid operon. Hence, said microorganisms are useful for the synthesis of any compound being part of the colanic acid pathway such as GDP-fucose, GDP-mannose and colanic acid, and/or, can be further used—starting form GDP-fucose as a precursor—to synthesize fucosylated oligosaccharides or—starting from GDP-mannose as a precursor—to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding for the transcriptional regulators ArcA and IclR lead to an acid resistance phenotype in the exponential growth phase allowing the synthesis of pH sensitive molecules or organic acids.

FIELD OF THE INVENTION

The present invention relates to mutated and/or transformedmicroorganisms for the synthesis of various compounds. Morespecifically, the present invention discloses microorganisms mutated inthe genes encoding for the regulators ArcA and IclR. The lattermutations result in a significant upregulation of the genes that arepart of the colanic acid operon. Hence, said microorganisms are usefulfor the synthesis of any compound being part of the colanic acid pathwaysuch as GDP-fucose, GDP-mannose and colanic acid, and/or, can be furtherused—starting from GDP-fucose as a precursor—to synthesize fucosylatedoligosaccharides or—starting from GDP-mannose as a precursor—tosynthesize mannosylated oligosaccharides. In addition, mutations in thegenes coding for the transcriptional regulators ArcA and IclR lead to anacid resistance phenotype in the exponential growth phase allowing thesynthesis of pH sensitive molecules and organic acids.

BACKGROUND OF THE INVENTION

The genes arcA encoding for the aerobic respiration control protein andiclR encoding the isocitrate lyase regulator are known to regulate thecentral carbon metabolism. ArcA is a global transcriptional regulatorthat regulates a wide variety of genes, while IclR is a localtranscriptional regulator that regulates the glyoxylate pathway. ArcA isknown to regulate the central carbon metabolism in response to oxygendeprivation and has no connection with IclR other than that it alsoregulates the glyoxylate pathway (24, 28, 29, 37, 38). In an earlierstudy the combined effect of ΔiclRΔarcA mutant strains on the centralcarbon metabolism has been observed. Increased fluxes were shown in thetricarboxylic acid (TCA) cycle and glyoxylate pathway and an interestingand surprising phenotype appeared when both genes where knocked out,namely the double mutant strain formed biomass with a yield thatapproached the maximal theoretical yield (4, 39).

Some compounds, such as GDP-fucose, are in high demand. The lattercompound is indeed a precursor of fucosylated oligosaccharides such asfucosyllactose, fucosyllactoNbiose and lewis X oligosaccharides, or, offucosylated proteins. These sugars are components of human mother milkin which they have anti-inflammatory and prebiotic effects and/or haveapplications in therapeutics as nutraceutical, anti-inflammatory agentor prebiotic, in addition, fucosylated proteins find applications in thepharmaceutics (5, 8, 27). However, an efficient method to produce thelatter high-value compounds is still needed.

In addition GDP-mannose is also an intermediate of the pathway towardsGDP-fucose. Interrupting the pathway prematurely leads to theaccumulation of this compound, which is a precursor of mannosylatedoligosaccharides. These oligosaccharides find for example applicationsin the treatment of gram-negative bacterial infections, in addition,GDP-mannose is important for the humanization of protein glycosylations,which is essential for the production of certain therapeutic proteins(18, 30). Mannosylated oligosaccharides and mannosylated glycoconjugatesare also used for drug targeting, for instance mannosylated antiviralscan specifically target the liver and kidneys (7).

The present invention provides microorganisms which are geneticallychanged in such a manner that they can efficiently produce the lattercompounds.

Moreover, the synthesis of pH sensitive molecules, such as—but notlimited to—glucosamine, and organic acids, such as—but not limitedto—pyruvic acid, succinic acid, adipic, sialic acid, sialylatedoligosaccharides . . . are preferably produced at low pH, either tostabilize the product or for downstream processing reasons (4, 12, 40).Therefore, strains that can grow at low pH are beneficial for theseproduction processes. E. coli is an organism that can adapt easily tovarious conditions, for instance it can easily adapt to the harsh pHconditions in the stomach, which is about pH 2 (14). Nonetheless, E.coli does not seem to grow at these conditions, but induces its acidresistance mechanisms in the stationary phase (40). During this phasethe cell does not multiply anymore and therefore hampers productivity.Up to now, no solution was found to this problem. However, in thepresent invention, a genetically engineered microorganism is providedthat can induce acid resistance mechanisms in the exponential growthphase, which is the phase that is mostly used for production of organicacids and pH instable products.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Relative gene expression pattern of the wild type, the ΔiclR andΔarcA mutant strain to the ΔarcAΔiclR mutant strain of genes involved incolanic acid biosynthesis in batch fermentation conditions. The genesinvolved in colanic acid biosynthesis are presented in FIGS. 3 and 4.

FIG. 2: Gene expression pattern of the colanic acid operon of the wildtype, the ΔiclR and ΔarcA mutant strain in chemostat fermentationconditions relative to the ΔarcAΔiclR mutant strain.

FIG. 3: The gene organisation of the colanic acid operon and an overviewof the function of these genes:

Gene: Function: wza Component of capsular polysaccharide exportapparatus wzb Tyrosine phosphatase wzc Tyrosine kinase wcaAGlycosyltransferase wcaB Acyltransferase wcaC Glycosyltransferase wcaDColanic acid polymerase wcaE Glycosyltransferase wcaF Acyltransferasegmd GDP-mannose-4,6-dehydratase fcl GDP-fucose synthase gmm GDP-mannosehydrolase wcal Glycosyltransferase cpsB Mannose-1-phosphateguanylyltransferase cpsG Phosphomannomutase wcaJ UDP-glucose lipidcarrier transferase wzxC Putative transporter wcaK PyruvyltransferasewcaL Glycosyltransferase wcaM Predicted protein in colanic acidbiosynthesis

FIG. 4: The colanic acid biosynthesis pathway.

FIG. 5: Regulatory network of the colanic acid operon. This network wasconstructed with Pathway tools v 13.0.

FIG. 6: Effect of the ΔarcAΔiclR mutations on the GDP-fucosebiosynthesis route.

FIG. 7: Overview of the genetic modifications needed to enhancefucosyllactose and fucosylated oligosaccharides production starting fromglucose as a substrate.

FIG. 8: Starting from sucrose, fucosylated sugar derivates such asfucosyllactose and more specifically 1,2-fucosyllactose are produced.The strain is modified to force the cell to produce fructose-6-phosphatewhich is an intermediate in the synthesis of GDP-fucose. Glucose orglucose-1-phosphate (if the starting enzyme is either a sucrase or asucrose phosphorylase) is then fed to the central carbon metabolism viathe pentose phosphate pathway.

FIG. 9: Overview of the genetic modifications needed to enhancefucosyllactose and fucosylated oligosaccharides production starting fromglucose as a substrate in a split metabolism.

FIG. 10: Detail of the pentose phosphate pathway flux in a strain inwhich the genes coding for phosphoglucose isomerase andphosphofructokinase are knocked out.

FIG. 11: Starting from sucrose, mannosylated sugar derivates areproduced. The strain is modified to force the cell to producefructose-6-phosphate which is an intermediate in the synthesis ofGDP-fucose. Glucose or glucose-1-phosphate (if the starting enzyme iseither a sucrase or a sucrose phosphorylase) is then fed to the centralcarbon metabolism via the pentose phosphate pathway.

FIG. 12: Gene expression pattern acid resistance related genes of thewild type, the ΔiclR and ΔarcA mutant strain in batch culturingconditions relative to the ΔarcAΔiclR mutant strain.

FIG. 13: LC MSMS analysis chromatograms of culture broth and a2-fucosyllactose standard. A. LC MSMS analysis of the standard, B. LCMSMS analysis of a sample of the culture broth of a mutant strainexpressing a H. pylori fucosyltransferase, C. LC MSMS analysis of asample of the culture broth of a mutant strain expressing a H. pylorifucosyltransferase.

FIG. 14: LC MSMS analysis mass spectrum from the chromatograms shown inFIG. 13 of culture broth and a 2-fucosyllactose standard. A. Mass (m/z)of the standard, B. Mass (m/z) of the sample of the culturing broth of amutant strain expressing a H. pylori fucosyltransferase, C. Mass (m/z)of the sample of the culturing broth of a mutant strain expressing a H.pylori fucosyltransferase.

FIG. 15: The sequence of the artificial hybrid promoter as given by SEQID No 6 (the combination of the native and an artificial promoter) thatwas cloned in front of the colanic acid operon.

DESCRIPTION OF INVENTION

The present invention provides microorganisms such as Enterobacteriaceaewhich are genetically changed in such a manner that they can efficientlyproduce compounds which are part of the colanic acid pathway. Aparticular compound of interest is GDP-fucose which is used as aprecursor to synthesize fucosylated oligosaccharides. The latter havehealth-promoting effects as indicated above but there is no efficientproduction method available to produce said compounds.

The present invention thus provides for the usage of a mutated and/ortransformed microorganism comprising a genetic change leading to amodified expression and/or activity of the transcriptional regulatorsthe aerobic respiration control protein ArcA and the isocitrate lyaseregulator IclR to upregulate at least one of the genes of the colanicacid operon, wherein said operon comprises the genes cpsG, cpsB, gmd andfcl that code for a phosphomannomutase, a mannose-1-phosphateguanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucosesynthase, respectively. The latter operon may also comprise the genescpsG, cpsB, gmd, fcl and wza. In addition the expression of the genercsA is increased. This gene is a transcriptional regulator of thecolanic acid operon. Enhanced expression of this gene increasestranscription of the colanic acid operon (13, 36).

Hence the present invention relates to the usage of a mutated and/ortransformed microorganism comprising a genetic change leading to amodified expression and/or activity of the transcriptional regulator,the aerobic respiration control protein, ArcA and the isocitrate lyaseregulator IclR to upregulate the transcriptional regulator of thecolanic acid operon, rcsA, which in turn upregulates at least one of thegenes of the colanic acid operon.

Hence, the present invention relates to a mutated and/or transformedmicroorganism such as—but not limited to Enterobacteriaceae such as anEscherichia coli (E. coli) strain comprising a genetic change leading toa modified expression of the transcriptional regulators: the aerobicrespiration control protein ArcA and the isocitrate lyase regulatorIclR.

A mutated and/or transformed microorganism such as E. coli as used herecan be obtained by any method known to the person skilled in the art,including but not limited to UV mutagenesis and chemical mutagenesis. Apreferred manner to obtain the latter microorganism is by disrupting(knocking-out) the genes (arcA and iclR) encoding for the proteins ArcAand IclR, or, by replacing the endogenous promoters of said genes byartificial promoters or replacing the endogenous ribosome binding siteby an artificial ribosome binding site. The term ‘artificial promoters’relates to heterologous or non-natural or in silico designed promoterswith known expression strength, these promoters can be derived fromlibraries as described by Alper et al. (2005), Hammer et al. (2006), orDe Mey et al. (2007) (3, 11, 15). The term heterologous promoter refersto any promoter that does not naturally occur in front of the gene. Theterm ‘artificial promoter’ may also refer to promoters with DNAsequences that are combinations of the native (autologous) promotersequence with parts of different (autologous or heterologous) promotersequences as for example shown further in the examples. Sequences ofsuch ‘artificial promoters’ can be found in databases such as forexample partsregistry.org (6). The term ‘artificial ribosome bindingsite’ relates to heterologous or non-natural or in silico designedribosome binding sites with known or measurable translation rates, theselibraries can be derived from libraries or designed via algorithms asdescribed by Salis et al (2009) (26). Hence, the present inventionspecifically relates to a mutated and/or transformed microorganism asindicated above wherein said genetic change is disrupting the genesencoding for ArcA and IclR, or, reducing or eliminating the function ofArcA and IclR via mutations in the coding sequence of the genes codingfor ArcA and IclR, or, is replacing the endogenous promoters of thegenes encoding for ArcA and IclR by artificial promoters; or, isreplacing the endogenous ribosome binding site by an artificial ribosomebinding site. It is further clear that the mutant and/or transformantaccording to the present invention may further comprise additionalgenetic changes in one or more other genes within its genome as is alsodescribed further. The term microorganism specifically relates to abacterium, more specifically a bacterium belonging to the family ofEnterobacteriaceae. The latter bacterium preferably relates to anystrain belonging to the species Escherichia coli such as but not limitedto Escherichia coli B, Escherichia coli C, Escherichia coli W,Escherichia coli K12, Escherichia coli Nissle. More specifically, thelatter term relates to cultivated Escherichia coli strains—designated asE. coli K12 strains—which are well-adapted to the laboratoryenvironment, and, unlike wild type strains, have lost their ability tothrive in the intestine. Well-known examples of the E. coli K12 strainsare K12 Wild type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100,JM101, NZN111 and AA200. Hence, the present invention specificallyrelates to a mutated and/or transformed Escherichia coli strain asindicated above wherein said E. coli strain is a K12 strain. Morespecifically, the present invention relates to a mutated and/ortransformed Escherichia coli strain as indicated above wherein said K12strain is E. coli MG1655.

The terms ‘leading to a modified expression or activity’ indicates thatthe above described mutations/transformations affects the transcriptionand/or translation and/or post-translational modification of said genes(arcA and iclR) into the transcriptional regulator proteins of thepresent invention (ArcA and IclR) in such a way that the lattertranscription has significantly decreased or has even been completelyabolished compared to a wild type strain, which has not been mutated ortransformed with regard to both particular genes of the presentinvention. Hence, the present invention relates to a mutated and/ortransformed microorganism such as an Escherichia coli strain asindicated above wherein said modified expression is a decreasedexpression, and, to a mutated and/or transformed microorganism such asan Escherichia coli strain as indicated above wherein said decreasedexpression is an abolished expression.

The terms ‘upregulating at least one of the genes of the colanic acidoperon’ indicates that the expression of at least 1, 2, 3, 4, . . . , orall of the genes belonging to the colanic acid operon are significantly(=P>0.05) upregulated when compared to the expression of said geneswithin a corresponding wild type microorganism which is cultivated underthe same conditions as the mutated and/or transformed microorganism. Thegenes which belong to the colanic acid operon are wza, wzb, wzc, wcaA,wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, cpsB, cpsG, wcaJ,wzxC, wcaK, wcaL and wcaM as indicated in FIG. 3 and/or as described in(35). Furthermore, the gene rcsA, coding for the transcriptionalregulator of the colanic acid operon is upregulated (13, 36). Morespecifically the terms ‘upregulating at least one of the genes of thecolanic acid operon’ or the transcriptional regulator of the colanicacid operon indicates that at least one of the genes of the colanic acidoperon is 6 to 8 times upregulated in comparison to the expression ofthe genes of the colanic acid operon in the corresponding wild typemicroorganism. In addition the present invention relates to upregulatinggenes of the colanic acid operon as described above by replacing thenative promoter by an ‘artificial promoter’. More specifically, thepresent invention relates to a combination of the sequence of the nativepromoter with sequences of other artificial promoter sequences. Thecombination of the sequence of the native promoter with the sequence ofother artificial promoter sequences is more specifically the replacementof the sigma factor binding site of the native promoter with a strongersigma factor binding site. Sigma factors, such as but not limited tosigma70, sigmaS, sigma24, . . . , are described (41), subunits of RNApolymerase that determine the affinity for promoter sequences and thetranscription rate of genes. The present invention providesmicroorganisms which are genetically changed in such a manner that theycan efficiently produce compounds which are part of the colanic acidpathway. The terms ‘compounds which are part of the colanic acidpathway’ refer to all compounds as indicated on FIG. 4 starting fromfructose-6-P and resulting in extracellular colanic acid. Morespecifically the latter terms refer to the compounds mannose-6-P,mannose-1-P, GDP-mannose, GDP-4-dehydro-6deoxy-mannose, GDP-fucose andcolanic acid. Hence the present invention specifically relates to theusage as indicated for the synthesis of colanic acid and/or for thesynthesis of GDP-fucose. As GDP-fucose is a precursor for fucosylatedoligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis Xoligosaccharide or fucosylated proteins, and as these sugars havetherapeutical, nutraceutical, anti-inflammatory and prebiotic effects,the present invention specifically relates to the usage as describedabove for the synthesis of fucosylated oligosaccharides. In other words,the present invention relates to a process for the synthesis of colanicacid and/or GDP-fucose and/or fucosylated oligosaccharides comprisinggenetically changing the transcriptional regulators the aerobicrespiration control protein ArcA and the isocitrate lyase regulator IclRto upregulate at least one of the genes of the colanic acid operon,wherein said operon comprises the genes cpsG, cpsB, gmd and fcl or genescpsG, cpsB, gmd, fcl and wza. More specifically, the present inventionrelates to a process as described wherein the mutations for ArcA andIclR are applied in combination with at least one mutation that enhancesthe production of fucosylated compounds. In order to efficiently producefucosylated oligosaccharides (see FIGS. 1, 2 and 5-10), the abovedescribed mutations in arcA and iclR can be applied in combination withother mutations which further enhance the production of fucosylatedcompounds. Some of these—non-limiting—other mutations are: a) thedeletion of wcaJ from the colanic operon, stopping the initiation of thecolanic acid biosynthesis and thus allowing the accumulation ofGDP-fucose; b) the introduction of a fucosyltransferase to link fucosewith different acceptor molecules such as lactose; c) for theaccumulation of the precursor of the GDP-fucose biosynthetic pathway andadditional to the deletion of wcaJ, at least one of the followingcolanic acid operon genes that do not code for GDP-fucose biosynthesisis knocked out: gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ,wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM; d) for the production offucosyllactose, lacZ coding for β-galactosidase, is knocked out to avoidlactose degradation; e) to accumulate the precursor fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced; f) because fructose-6-phosphate is easily degraded in theglycolysis, the glycolysis has to be interrupted in order to steer allfructose-6-phosphate in the direction of GDP-fucose and the genes pgi,pfkA and pfkB (coding for glucose-6-phosphate isomerase andphosphofructokinase A and B) are thus knocked out; g) reducing proteindegradation by knocking out a protease coded by a gene such as Ion; h)By constitutively expressing a lactose permease, subpopulations areavoided in the production process which are common for lactose inducedgene expression systems (19). In other words, the present inventionrelates to a process as described above for the synthesis of fucosylatedoligosaccharides wherein said at least one mutation that enhance theproduction of fucosylated compounds is: the deletion of the wcaJ gene,and/or, knocking-out the colanic acid operon genes gmm, wcaA, wcaB,wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc,and/or, wcaM, and/or, knocking-out lacZ, and/or, introducing a sucrosephosphorylase or invertase, and/or, knocking out the genes pgi, pfkA andpfkB, and/or, knocking out the gene Ion, and/or introducing afucosyltransferase, and/or a lactose permease. The term ‘introducing afucosyltransferase’ relates to upregulating or heterologous expressionof fucosyltransferases which are within, but not limited to the enzymesin enzyme classes EC2.4.1.65, 2.4.1.68, 2.4.1.69, 2.4.1.152, 2.4.1.214,and/or 2.4.1.221 and/or the glycosyltransferase families GT1, GT2, GT10GT11, GT23, GT37, GT65, GT68, and/or GT74 and/or originating from butnot limited to Helicobacter pylori, Campylobacter jejuni, Dictyostelliumdiscoideum, Mus musculus, Homo sapiens, . . . and thesefucosyltransferases catalyse the formation of α(1,2), α(1,3), α(1,4), orα(1,6) bonds on other sugars such as but not limited to galactose,lactose, lactoNbiose, lactoNtetraose, lactosamine, lactoNtetraose,sialyllactoses, disialyllactoses, or fucosylated proteins, orfucosylated fatty acids, or fucosylated aglycons such as, but notlimited to, antivirals, antibiotics, . . . .

The present invention provides for the usage of a mutated and/ortransformed microorganism comprising a genetic change leading to amodified expression and/or activity of the transcriptional regulatorsthe aerobic respiration control protein ArcA and the isocitrate lyaseregulator IclR to upregulate at least one of the genes of the colanicacid operon, wherein said operon comprises the genes cpsG and cpsB,coding for phosphomannomutase and mannose-1-phosphateguanylyltransferase, which are needed for the biosynthesis ofGDP-mannose. As GDP-mannose is a precursor for mannosylatedoligosaccharides and mannosylated glycoconjugates. Theseoligosaccharides and glycoconjugates find for example applications inthe treatment of gram-negative bacterial infections, in addition,GDP-mannose is important for the humanization of protein glycosylations,which is essential for the production of certain therapeutic proteins(18, 30). Mannosylated oligosaccharides and mannosylated glycoconjugatesare also used for drug targeting, for instance mannosylated antiviralscan specifically target the liver and kidneys (7). In order toefficiently produce mannosylated oligosaccharides (see FIGS. 1, 2, 5, 6and 11), the above described mutations in arcA and iclR can be appliedin combination with other mutations which further enhance the productionof mannosylated compounds. Some of these—non-limiting—other mutationsare: a) the gene gmd of the colanic acid operon is deleted, and/or, b)wherein the gene gmm coding for GDP-mannose hydrolase is deleted,and/or, c) wherein the colanic acid operon genes that do not code forGDP-mannose biosynthesis reactions, the genes gmm, wcaA, wcaB, wcaC,wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, fcl, gmd, wzx, wza, wzband/or, wcaM, are deleted, and/or, d) wherein a gene encoding for asucrose phosphorylase or an invertase is introduced, and/or, e) whereinthe genes pgi, pfkA and pfkB, coding for phosphoglucose isomerase,phosphofructokinase A and phosphofructokinase B respectively, aredeleted, and/or, f) knocking out the gene Ion encoding for a protease,and/or f) wherein a gene encoding for a mannosyltransferase isintroduced. In other words, the present invention relates to a processas described above for the synthesis of colanic acid and/or GDP-fucoseand/or fucosylated oligosaccharides for the synthesis of GDP-mannoseand/or for the synthesis of mannosylated oligosaccharides. The presentinvention further relates to said process wherein the genes cpsG andcpsB of the colanic acid operon are upregulated and wherein: a) the genegmd of the colanic acid operon is deleted, and/or, b) wherein the genegmm is deleted, and/or c) wherein the colanic acid operon genes fcl,gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL,wzx, wza, wzb, wzc, and/or, wcaM are knocked out and/or, d) wherein agene encoding for a sucrose phosphorylase or an invertase is introduced,and/or, e) wherein the genes pgi, pfkA and pfkB are deleted, and/or, f)knocking out the gene Ion, and/or g) wherein a gene encoding for amannosyltransferase is introduced. The term ‘introducing amannosyltransferase’ relates to upregulating or heterologous expressionof mannosyltransferases which are within, but not limited to the enzymesin enzyme classes EC 2.4.1.32, 2.4.1.627, 2.4.1.644, 2.4.1.48, 2.4.1.54,2.4.1.57, 2.4.1.83, 2.4.1.109, 2.4.1.110, 2.4.1.119, 2.4.1.130,2.4.1.131, 2.4.1.132, 2.4.1.142, 2.4.1.199, 2.4.1.217, 2.4.1.232,2.4.1.246, 2.4.1.251, 2.4.1.252, 2.4.1.257, 2.4.1.258, 2.4.1.259,2.4.1.260, 2.4.1.265, and/or 2.4.1.270 and/or the glycosyltransferasefamilies GT1, GT2, GT4, GT15, GT22, GT32, GT33, GT39, GT50 and/or GT58and/or originating from but not limited to Helicobacter pylori,Campylobacter jejuni, Dictyostellium discoideum, Mus musculus, Homosapiens, . . . and these mannosyltransferases catalyse the formation ofα(1,2), α(1,3), α(1,4), or α(1,6) bonds on other sugars such as but notlimited to galactose, N-acetylglucosamine, Rhamnose, lactose,lactoNbiose, lactoNtetraose, lactosamine, lactoNtetraose,sialyllactoses, disialyllactoses, or mannosylated proteins, ormannosylated fatty acids, or mannosylated aglycons such as, but notlimited to, antivirals, antibiotics, . . . .

The term ‘heterologous expression’ relates to the expression of genesthat are not naturally present in the production host, genes which canbe synthesized chemically or be picked up from their natural host viaPCR, genes which can be codon optimized for the production host or inwhich point mutation can be added to enhance enzyme activity orexpression. Expressing heterologous and/or native genes can either bedone on the chromosome, artificial chromosomes or plasmids andtranscription can be controlled via inducible, constitutive, native orartificial promoters and translation can be controlled via native orartificial ribosome binding sites.

Consequently, the present invention further relates to mutated and/ortransformed organisms in which the regulators ArcA and IclR as describeabove, in combination with the genes encoding for the enzymesphosphoglucose isomerase and phosphofructokinase, are knocked out or arerendered less functional. More specifically, the present inventionrelates to the latter organisms wherein the enzyme phosphoglucoseisomerase is encoded by the gene pgi and wherein the enzymephosphofructokinase is encoded by the gene(s) pfkA and/or pfkB.

The terms ‘genes which are rendered less-functional or non-functional’refer to the well-known technologies for a skilled person such as theusage of siRNA, RNAi, miRNA, asRNA, mutating genes, knocking-out genes,transposon mutagenesis, etc. . . . which are used to change the genes insuch a way that they are less able (i.e. statistically significantly‘less able’ compared to a functional wild-type gene) or completelyunable (such as knocked-out genes) to produce functional final products.The term ‘(gene) knock out’ thus refers to a gene which is renderednon-functional. The term ‘deleted gene’ or ‘gene deletion’ also refersto a gene which is rendered non-functional.

The present invention further relates to a mutated and/or transformedorganism as described in the latter paragraph wherein said organism isfurther transformed with a gene encoding for a sucrose phosphorylase.

The present invention also relates to a mutated and/or transformedorganism as described above wherein, in addition, the activity and/orexpression of the gene encoding for a lactose permease is madeconstitutive and/or increased. Said activity can be increased byover-expressing said gene and/or by transforming said organisms with agene encoding for a lactose permease.

The present invention further relates to any mutated and/or transformedorganism as described above wherein at least one of the following genesis knocked out or is rendered less functional:

a gene encoding for a beta-galactosidase, a gene encoding for aglucose-1-phosphate adenylyltransferase, a gene encoding for aglucose-1-phosphatase, a gene encoding for phosphogluconate dehydratase,a gene encoding for 2-keto-3-deoxygluconate-6-phosphate aldolase, a geneencoding for a glucose-1-phosphate uridyltransferase, a gene encodingfor an UDP-glucose-4-epimerase, a gene encoding for anUDP-glucose:galactose-1-phosphate uridyltransferase, a gene encoding foran UDP-galactopyranose mutase, a gene encoding for anUDP-galactose:(glucosyl)lipopolysaccharide-1,6-galactosyltransferase, agene encoding for an UDP-galactosyltransferase, a gene encoding for anUDP-glucosyltransferase, a gene encoding for an UDP-glucuronatetransferase, a gene encoding for an UDP-glucose lipid carriertransferase, a gene encoding for a GDP-mannose hydrolase, a geneencoding for an UDP-sugar hydrolase, a gene encoding for amannose-6-phosphate isomerase, a gene encoding for anUDP-N-acetylglucosamine enoylpyruvoyl transferase, a gene encoding foran UDP-N-acetylglucosamine acetyltransferase, a gene encoding for anUDP-Nacetylglucosamine-2-epimerase, a gene encoding for anundecaprenyl-phosphate alfa-N-acetylglucosaminyl transferase, a geneencoding for a glucose-6-phosphate-1-dehydrogenase, and/or, a geneencoding for a L-glutamine:D-fructose-6-phosphate aminotransferase, agene encoding for a mannose-6-phosphate isomerase, a gene encoding for asorbitol-6-phosphate dehydrogenase, a gene encoding for amannitol-1-phosphate 5-dehydrogenase, a gene encoding for aallulose-6-phosphate 3-epimerase, a gene encoding for an invertase, agene encoding for a maltase, a gene encoding for a trehalase, a geneencoding for a sugar transporting phosphotransferase, a gene encodingfor a protease, or a gene encoding for a hexokinase. The term ‘at leastone’ indicated that at least 1, but also 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32 or all 33 genes is (are) knocked out or is (are) renderedless functional.

The present invention further relates also to the usage of a mutatedand/or transformed microorganism such as an Escherichia coli straincomprising a genetic change leading to a modified expression of thetranscriptional regulators the aerobic respiration control protein ArcAand the isocitrate lyase regulator IclR to upregulate at least one ofthe following acid resistance related genes: ydeP, ydeO, hdeA, hdeD,gadB, gadC, gadE, gadX, gadW and/or slp (17, 22). These genes arenormally expressed in stationary phase conditions; however, the presentmutated and/or transformed microorganism is able to enhance theexpression of these acid resistance related genes in the exponentialgrowth phase. Hence, the present invention relates to the usage asdescribed above for the synthesis of acids or pH sensitive moleculessuch as but not limited to glucosamine which is pH sensitive and shouldbe produced at low pH (12). Organic acids, such as but not limited topyruvic acid, succinic acid, adipic, sialic acid, sialylatedoligosaccharides (e.g. sialyllactose, sialyl Lewis X sugars, . . . ),acetylated oligosaccharides (chitins, chitosans, . . . ), sulfonatedoligosaccharides (heparans and heparosans) . . . are preferably producedat low pH for downstream processing purposes (4). In other words, thepresent invention relates to a process for the synthesis of acids,sialic acid, sialylated oligosaccharides or glucosamine comprisinggenetically changing the transcriptional regulators the aerobicrespiration control protein ArcA and the isocitrate lyase regulator IclRto upregulate at least one of the following acid resistance relatedgenes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp.

The present invention will now be illustrated by the followingnon-limiting examples.

EXAMPLES

A high throughput RT-qPCR screening of the microorganisms of the presentinvention has been setup with Biotrove OpenArray® technology. In thisexperiment the transcription of 1800 genes were measured in 4 strains(wild type, ΔarcA, ΔiclR, ΔarcA ΔiclR) in two conditions (chemostat andbatch). The data was processed using a curve fitting toolbox in R (25,34) and Quantile Normalization, the error on the data was calculatedusing Bayesian statistics (20, 21, 31).

Material and Methods Strains and Plasmids

Escherichia coli MG1655 [⁻, F⁻, rph-1] was obtained from the NetherlandsCulture Collection of Bacteria (NCCB). Escherichia coli BL21(DE3) wasobtained from Novagen. Escherichia coli MG1655 ackA-pta, poxB, pppcppc-p37 (10), the single knock-outs E. coli MG1655 arcA and E. coliMG1655 iclR and the double knock-out E. coli MG1655 arcA, iclR wereconstructed in the Laboratory of Genetics and Microbiology (MICR) usingthe method of Datsenko & Wanner (9).

Media

The Luria Broth (LB) medium consisted of 1% tryptone peptone (Difco,Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodiumchloride (VWR, Leuven, Belgium). Shake flask medium contained 2 g/lNH₄Cl, 5 g/l (NH₄)₂SO₄, 2.993 g/l KH₂PO₄, 7.315 g/l K₂HPO₄, 8.372 g/lMOPS, 0.5 g/l NaCl, 0.5 g/l MgSO₄.7H₂O, 16.5 g/l glucose.H₂O, 1 ml/lvitamin solution, 100 μl/l molybdate solution, and 1 ml/l seleniumsolution. The medium was set to a pH of 7 with 1M KOH.

Vitamin solution consisted of 3.6 g/l FeCl₂.4H₂O, 5 g/l CaCl₂.2H₂O, 1.3g/l MnCl₂. 2H₂O, 0.38 g/l CuCl₂.2H₂O, 0.5 g/l CoCl₂.6H₂O, 0.94 g/lZnCl₂, 0.0311 g/l H₃BO₄, 0.4 g/l Na₂EDTA.2H₂O and 1.01 g/l thiamine.HCl.The molybdate solution contained 0.967 g/l Na₂MoO₄.2H₂O. The seleniumsolution contained 42 g/l SeO₂.

The minimal medium for fermentations contained 6.75 g/l NH₄Cl, 1.25 g/l(NH₄)₂SO₄, 1.15 g/l KH₂PO₄, 0.5 g/l NaCl, 0.5 g/l MgSO₄.7H₂O, 16.5 g/lglucose.H₂O, 1 ml/l vitamin solution, 100 μl/l molybdate solution, and 1ml/l selenium solution with the same composition as described above.

Cultivation Conditions

A preculture, from a single colony on a LB-plate, in 5 ml LB medium wasincubated during 8 hours at 37° C. on an orbital shaker at 200 rpm. Fromthis culture, 2 ml was transferred to 100 ml minimal medium in a 500 mlshake flask and incubated for 16 hours at 37° C. on an orbital shaker at200 rpm. 4% inoculum was used in a 2 l Biostat B Plus culture vesselwith 1.5 l working volume (Sartorius Stedim Biotech, Melsungen,Germany). The culture conditions were: 37° C., stirring at 800 rpm, anda gas flow rate of 1.5 l/min. Aerobic conditions were maintained bysparging with air, anaerobic conditions were obtained by flushing theculture with a mixture of 3% CO₂ and 97% of N₂. The pH was maintained at7 with 0.5 M H₂SO4 and 4 M KOH. The exhaust gas was cooled down to 4° C.by an exhaust cooler (Frigomix 1000, Sartorius Stedim Biotech,Melsungen, Germany). 10% solution of silicone antifoaming agent (BDH331512K, VWR Int Ltd., Poole, England) was added when foaming raisedduring the fermentation (approximately 10 μl). The off-gas was measuredwith an EL3020 off-gas analyser (ABB Automation GmbH, 60488 Frankfurt amMain, Germany).

All data was logged with the Sartorius MFCS/win v3.0 system (SartoriusStedim Biotech, Melsungen, Germany).

All strains were cultivated at least twice and the given standarddeviations on yields and rates are based on at least 10 data pointstaken during the repeated experiments.

Sampling Methodology

The bioreactor contains in its interior a harvest pipe (BD SpinalNeedle, 1.2×152 mm (BDMedical Systems, Franklin Lakes, N.J.—USA)connected to a reactor port, linked outside to a Masterflex-14 tubing(Cole-Parmer, Antwerpen, Belgium) followed by a harvest port with aseptum for sampling. The other side of this harvest port is connectedback to the reactor vessel with a Masterflex-16 tubing. This system isreferred to as rapid sampling loop. During sampling, reactor broth ispumped around in the sampling loop. It has been estimated that, at aflow rate of 150 ml/min, the reactor broth needs 0.04 s to reach theharvest port and 3.2 s to re-enter the reactor. At a pO2 level of 50%,there is around 3 mg/l of oxygen in the liquid at 37° C. The pO2 levelshould never drop below 20% to avoid micro-aerobic conditions. Thus 1.8mg/l of oxygen may be consumed during transit through the harvestingloop. Assuming an oxygen uptake rate of 0.4 g oxygen/g biomass/h (themaximal oxygen uptake rate found at μ_(max)), this gives for 5 g/lbiomass, an oxygen uptake rate of 2 g/l/h or 0.56 mg/l/s, whichmultiplied by 3.2 s (residence time in the loop) gives 1.8 mg/l oxygenconsumption.

In order to quench the metabolism of cells during the sampling, reactorbroth was sucked through the harvest port in a syringe filled with 62 gstainless steel beads pre-cooled at −20° C., to cool down 5 ml brothimmediately to 4° C. Sampling was immediately followed by coldcentrifugation (15000 g, 5 min, 4° C.). During the batch experiments, asample for OD_(600nm) and RT-qPCR measurements was taken using the rapidsampling loop and the cold stainless bead sampling method.

RT-qPCR

mRNA was extracted with the RNeasy kit (Qiagen, Venlo, The Netherlands).RNA quality and quantity was checked with a nanodrop ND-1000spectrophotometer (Nanodrop technologies, Wilmingto, USA). The ratios260:280 (nm) and 260:230 (nm) were between 1.8 and 2 and at least 100ng/μl was needed for further analysis. cDNA was synthesised with randomprimers with the RevertAid™ H minus first strand cDNA synthesis kit(Fermentas, St. Leon-Rot, Germany). Finally, the gene expression of 1800genes was measured with the Biotrove OpenArray Real time PCR platform.The primers for the RT-PCR assay were designed with Primer design toolsfrom the Primer database (23).

The reaction mixture was composed as described in the BiotroveOpenArray™ Real-Time qPCR system users' manual. In short, a mastermixwas made with 26.4 μl LightCycler® DNA Master SYBR® Green I (Rocheapplied Science), 1.1 μl SYBR GREEN I (100× stock solution, SigmaS9430), 8.8 μl glycerol (Sigma G5150), 5.3 μl Pluronic® F68 (10% stock,Invitrogen), 2.64 μl BSA (Sigma A7906), 26.4 μl magnesium chloride (25mM stock solution, supplied in the LightCycler® kit of Roche appliedScience), 21.1 μl HiDi™ formamide (Applied biosystems), and 94.66 μlRNase free sterile water resulting in a 186.4 μl mastermix, which isenough to load 1 OpenArray™. For 1 SubArray (each OpenArray issubdivided in 48 SubArrays on which 1 sample can be loaded) 1.5 μlsample (with a concentration of 100 ng/μl) was mixed with 3.5 μl ofmastermind, as a no template control, water was used as blanc. Thesample-mastermix mixture was loaded in a Loader plate (MatriPlate™384-well black low volume polypropylene plate, Biotrove) in a RNase freehood. A full loader plate was loaded with an AutoLoader (Biotrove) andloader tips onto the OpenArrays. These OpenArrays were then submerged inOpenArray™ immersion fluid in an OpenArray™ Real-Time qPCR case. Thecase was sealed with Case sealing glue and incubated in the Case Sealingstation, which polymerizes the glue with UV light.

Analytical Methods

Cell density of the culture was frequently monitored by measuringoptical density at 600 nm (Uvikom 922 spectrophotometer, BRS, Brussel,Belgium). Cell dry weight was obtained by centrifugation (15 min, 5000g, GSA rotor, Sorvall RC-5B, Goffin Meyvis, Kapellen, Belgium) of 20 greactor broth in pre-dried and weighted falcons. The pellets weresubsequently washed once with 20 ml physiological solution (9 g/l NaCl)and dried at 70° C. to a constant weight. To be able to convertOD_(600nm) measurements to biomass concentrations, a correlation curveof the OD_(600nm) to the biomass concentration was made. Theconcentrations of glucose and organic acids were determined on a VarianProstar HPLC system (Varian, Sint-Katelijne-Waver, Belgium), using anAminex HPX-87H column (Bio-Rad, Eke, Belgium) heated at 65° C., equippedwith a 1 cm precolumn, using 5 mM H2SO4 (0.6 ml/min) as mobile phase. Adual-wave UV-VIS (210 nm and 265 nm) detector (Varian Prostar 325) and adifferential refractive index detector (Merck LaChrom L-7490, Merck,Leuven, Belgium) was used for peak detection. By dividing theabsorptions of the peaks in both 265 and 210 nm, the peaks could beidentified. The division results in a constant value, typical for acertain compound (formula of Beer-Lambert).

Glucose, fructose, sucrose, fucosyllactose and glucose-1-phosphate weremeasured by HPLC with a Hypercarb column and were detected with an MSMSdetector (Antonio et al., 2007; Nielsen et al., 2006).

Genetic Methods

All mutant strains were constructed via the methods described below.

Plasmids were maintained in the host E. coli DH5α (F⁻, φ80dlacZΔM15,Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk⁻, mk⁺), phoA, supE44,λ⁻, thi-1, gyrA96, relA1).

Plasmids.

pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains anFRT-flanked chloramphenicol resistance (cat) gene), pKD4 (contains anFRT-flanked kanamycin resistance (kan) gene), and pCP20 (expresses FLPrecombinase activity) plasmids were used for the mutant construction.The plasmid pBluescript (Fermentas, St. Leon-Rot, Germany) was used toconstruct the derivates of pKD3 and pKD4 with a promoter library, orwith alleles carrying a point mutation.

Mutations.

The mutations consisted in gene disruption (knock-out, KO). They wereintroduced using the concept of Datsenko and Wanner (9). The primers forthe mutation strategies are described in Table 1.

Transformants carrying a Red helper plasmid were grown in 10 ml LB mediawith ampicillin (100 mg/I) and L-arabinose (10 mM) at 30° C. to anOD_(600nm) of 0.6. The cells were made electrocompetent by washing themwith 50 ml of ice-cold water, a first time, and with 1 ml ice-coldwater, a second time. Then, the cells were resuspended in 50 μl ofice-cold water. Electroporation was done with 50 μl of cells and 10-100ng of linear double-stranded-DNA product by using a Gene Pulser™(BioRad) (600 Ω, 25 μFD, and 250 volts).

After electroporation, cells were added to 1 ml LB media incubated 1 hat 37° C., and finally spread onto LB-agar containing 25 mg/l ofchloramphenicol or 50 mg/l of kanamycin to select antibiotic resistanttransformants. The selected mutants were verified by PCR with primersupstream and downstream of the modified region and were grown in LB-agarat 42° C. for the loss of the helper plasmid. The mutants were testedfor ampicillin sensitivity.

Linear Double-Stranded-DNA.

The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 andtheir derivates as template. The primers used had a part of the sequencecomplementary to the template and another part complementary to the sideon the chromosomal DNA where the recombination has to take place (Table1). For the KO, the region of homology was designed 50-nt upstream and50-nt downstream of the start and stop codon of the gene of interest.For the KI, the transcriptional starting point (+1) had to be respected.PCR products were PCR-purified, digested with DpnI, repurified from anagarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).

Elimination of the Antibiotic Resistance Gene.

The selected mutants (chloramphenicol or kanamycin resistant) weretransformed with pCP20 plasmid, which is an ampicillin andchloramphenicol resistant plasmid that shows temperature-sensitivereplication and thermal induction of FLP synthesis. Theampicillin-resistant transformants were selected at 30° C., after whicha few were colony purified in LB at 42° C. and then tested for loss ofall antibiotic resistance and of the FLP helper plasmid. The gene knockouts and knock ins are checked with control primers (Fw/Rv-gene-out).These primers are given in Table 1.

TABLE 1Primers used to create E. coli MG1655 arcA, E. coli MG1655 iclR and thedouble knock-out E. coli MG1655 arc4, iclR and all other genetic knock outs andknock ins Primer name Sequence lacZ FW_LacZ_P1CATAATGGATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAgtgtaggctggagctgcttc (SEQ ID No 7) RV_LacZ_P2GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTcatatgaatatcctccttag (SEQ ID No 8) FW_LacZ_out GCGGTTGGAATAATAGCG (SEQ ID No 9)RV_LacZ_out CAGGTTTCCCGACTGGAAAG (SEQ ID No 10) glgC FW-glgC-P1Agaccgccggttttaagcagcgggaacatctctgaacatacatgtaaaacctgcagtgtaggctggagctgcttc (SEQ ID No 11) RV-glgC-P2Gtctggcagggacctgcacacggattgtgtgtgttccagagatgataaaaaaggagttagtccatatgaatatcctccttag (SEQ ID No 12) FW-glgC-outGcgaatatcgggaaatgcagg (SEQ ID No 13) RV-glgC-outCagagattgttttacctgctgg (SEQ ID No 14) agp FW_agp_P1CATATTTCTGTCACACTCTTTAGTGATTGATAACAAAAGAGGTGCCAGGAgtgtaggctggagctgcttc (SEQ ID No 15) RV_agp_P2TAAAAACGTTTAACCAGCGACTCCCCCGCTTCTCGCGGGGGAGTTTTCTGcatatgaatatcctccttag (SEQ ID No 16) FW_agp_out GCCACAGGTGCAATTATC (SEQ ID No 17)RV_agp_out CATTTTCGAAGTCGCCGGGTACG (SEQ ID No 18) Pgd Fw-pgi-P1GGCGCTACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCgtgtaggctggagctgcttc (SEQ ID No 19) Rv-pgi-P2GGTTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGcatatgaatatcctccttag (SEQ ID No 20) Fw_pgi_outGGCTCCTCCAACACCGTTAC (SEQ ID No 21) (2) Rv pgi outTACATATCGGCATCGACCTG (SEQ ID No 22) (2) pfkA Fw-pfkA-outTACCGCCATTTGGCCTGAC (SEQ ID No 23) Rv-pfkA-outAAAGTGCGCTTTGTCCATGC (SEQ ID No 24) Fw-pfkA-P1GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggctggagctgcttc (SEQ ID No 25) Rv-pfkA-P2GCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCcatatgaatatcctccttag (SEQ ID No 26) pfkB Fw-pfkB-outTAGCGTCCCTGGAAAGGTAAC (SEQ ID No 27) Rv-pfkB-outTCCCTCATCATCCGTCATAG (SEQ ID No 28) Fw-pfkB-P1CACTTTCCGCTGATTCGGTGCCAGACTGAAATCAGCCTATAGGAGGAAATGgtgtaggctggagctgcttc (SEQ ID No 29) Rv-pfkB-P2GTTGCCGACAGGTTGGTGATGATTCCCCCAATGCTGGGGGAATGTTTTTGcatatgaatatcctccttag (SEQ ID No 30) arcA FW-arcA-P1Ggttgaaaaataaaaacggcgctaaaaagcgccgttttttttgacggtggtaaagccgagtgtaggctggagctgcttc (SEQ ID No 31) RV-arcA-P2Ggtcagggacttttgtacttcctgtttcgatttagttggcaatttaggtagcaaaccatatgaatatcctccttag (SEQ ID No 32) FW-arcA-outCtgccgaaaatgaaagccagta (SEQ ID No 33) RV-arcA-outGgaaagtgcatcaagaacgcaa (SEQ ID No 34) iclR FW-iclR-P1Ttgccactcaggtatgatgggcagaatattgcctctgcccgccagaaaaaggtgtaggctggagctgcttc (SEQ ID No 35) RV-iclR-P2Gttcaacattaactcatcggatcagttcagtaactattgcattagctaacaataaaacatatgaatatcctccttag (SEQ ID No 36) FW-iclR-outCggtggaatgagatcttgcga (SEQ ID No 37) RV-iclR-outActtgctcccgacacgctca (SEQ ID No 38) FW_iclR_P8TTGCCACTCAGGTATGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGccgcttacagacaagctgtg (SEQ ID No 39) RV_iclR_P9GTTCAACATTAACTCATCGGATCAGTTCAGTAACTATTGCATTAGCTAACAATAAAAagccatgacccgggaattac (SEQ ID No 40) Rv-iclR-CTATTGCATTAGCTAACAATAAAACTTTTTCTGGCGGGCAGAGG (SEQ ID No 41) scarless KOstap 2 Fw-iclR-CCTCTGCCCGCCAGAAAAAGTTTTATTGTTAGCTAATGCAATAGTTAC (SEQ ID No 42)scarless KO stap 2 wcaJ Fw_wcaJ_out GCCAGCGCGATAATCACCAG (SEQ ID No 43)Rv_wcaJ_out TGCGCCTGAATGTGGAATC (SEQ ID No 44) Fw-wcaJ_2-TTTTGATATCGAACCAGACGCTCCATTCGCGGATGTACTCAAGGTCGAACgtgtaggct P1ggagctgcttc (SEQ ID No 45) Rv-wcaJ_2-TCTATGGTGCAACGCTTTTCAGATATCACCATCATGTTTGCCGGACTATGcatatgaat P2atcctccttag (SEQ ID No 46) fw_wcaJ_H1′_TCAATATGCCGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATATCGAACCAGACG (SEQ ID No 47) Rv_wcaJ_H2′_TGACAAATCTAAAAAAGCGCGAGCGAGCGAAAACCAATGCATCGTTAATCTCTATGGTGCAACGCTTTTC (SEQ ID No 48) Fw_wcaJ_H1′_CGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATATCGAACCAGA 2CGCTCCATTCG (SEQ ID No 49) Ion FW-Ion-P1CAGTCGTGTCATCTGATTACCTGGCGGAAATTAAACTAAGAGAGAGCTCTgtgtaggctggagctgcttc (SEQ ID No 50) oMEMO100 RV-CGAATTAGCCTGCCAGCCCTGTTTTTATTAGTGCATTTTGCGCGAGGTCAcatatgaat Ion-P2atcctccttag (SEQ ID No 51) oMEMO101 FW-AGCGCAACAGGCATCTGGTG (SEQ ID No 52) Ion-out oMEMO102 RV-TATATCAGGCCAGCCATCCC (SEQ ID No 53) Ion-out lacZYA:P22- lacY Fw_lacZYA_GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTCATTTA chlAATGGCGCGC (SEQ ID No 54) rv_lacZYA_GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTCGCCTACCT chlGTGACGGAAG (SEQ ID No 55) fw P221acY-GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTGTGTAGGCT KI_P1GGAGCTGCTTC (SEQ ID No 56) rv_P221acY-GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTAAGCGA KICTTCATTCACC (SEQ ID No 57) fw_lacZYA HCGACGCTTGTTCCTGCGCTTTGTTCATGCCGGATGCGGCTAATGTAGATCGCTGAACTT 1′GTAGGCCTG (SEQ ID No 58) rv_lacZYA HCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGC 2′AATTAATGTG (SEQ ID No 59) pfkA:P22- BaSP Fw-pfkA-P1GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggctggagctgcttc (SEQ ID No 60) Rv-pfkA-GCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCaagcttgcat pCXP22_P2gcctgcatcc (SEQ ID No 61) FW_kan AGAGGCTATTCGGCTATGAC (SEQ ID No 62)Fw_baSP_seq CGCCATGTTGGAATGGGAGG (SEQ ID No 63) Fw_pfkA_H1_TGATTGTTATACTATTTGCACATTCGTTGGATCACTTCGATGTGCAAGAAGACTTCCGG extCAACAGATTTC (SEQ ID No 64) Rv_pfkA_H2_AATTGCAGAATTCATGTAGGCCTGATAAGCGAAGCGCATCAGGCATTTTTGCTTCTGTC extATCGGTTTCAG (SEQ ID No 65) Fw-pfkA-outTACCGCCATTTGGCCTGAC (SEQ ID No 66) Rv-pfkA-outAAAGTGCGCTTTGTCCATGC (SEQ ID No 67) adhE:P22-frk Fw-adhE-ATCGGCATTGCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCTACTGAgtgtaggct pCXP22-P1ggagctgcttc (SEQ ID No 68) Rv-adhE-ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATTaagcttgca pCXP22-P2tgcctgcatcc (SEQ ID No 69) Fw-adhE-H1′AAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTTAACTGATCGGCATTGCCCAGAAG (SEQ ID No 70) Rv-adhE-H2′TTGATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCGTATTCGAGCAGATGATTTACTAAAAAAG (SEQ ID No 71) FW adhE outGCGTCAGGCAGTGTTGTATC (SEQ ID No 72) RV adhE outCTGGAAGTGACGCATTAGAG (SEQ ID No 73) ldhA:P14-FT_H. pylori FW_ldhA_outtgtcattacttacacatcccgc (SEQ ID No 74) RV_ldhA_outgcattcaatacgggtattgtgg (SEQ ID No 75) Fw-ldhA-CATTGGGGATTATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGgtgtaggct pCXP22_P1ggagctgcttc (SEQ ID No 76) Rv-ldhA-TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGaagcttgca pCXP22_P2tgcctgcatcc (SEQ ID No 77) Fw-ldhA-H1′CAATTACAGTTTCTGACTCAGGACTATTTTAAGAATAGAGGATGAAAGGTCATTGGGGATTATCTGAATCAG (SEQ ID No 78) Rv-ldhA-H2′GAATTTTTCAATATCGCCATAGCTTTCAATTAAATTTGAAATTTTGTAAAATATTTTTAGTAGCTTAAATGTGATTCAAC (SEQ ID No 79) Fw-ldhA-TTCACCGCTAAAGCGGTTAC (SEQ ID No 80) long homol Rv-ldhA-CGCGTAATGCGTGGGCTTTC (SEQ ID No 81) long homol promCA:P14 pCXP14_SP_CCGGCATATGGTATAATAGGG (SEQ ID No 82) Fw yegH_rc_ACGGCTTGCTGGCCATCA (SEQ ID No 83) pure_rv fw_P14-CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCTACGGCCCC CA_KI_tetAAAGGTCCAA (SEQ ID No 84) rv_P14-AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTATTGGCTTCA CA_KI_tetAGGGATGAGGCG (SEQ ID No 85) fw_P14- TCCCGACTACGTGGACCTTG (SEQ ID No 86)CA_KI_ overlapA ry P14-CATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGT CA_KI_CGACCTCGGCATTATTGGAAAGCCAATATTC (SEQ ID No 87) overlapA fw P14-GCCGCCATGGAAATTTCCCTATTATACCATATGCCGGCCAAGATGTCAAGAAACTTATA CA_KI_GAATGAAGTAAGTGTCATTCAATATGG (SEQ ID No 88) overlapB fw P14-AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTACTTCATTCT CA_KI_H1ATAAGTTTCTTGAC (SEQ ID No 89) rv_P14-CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCCGAGGTCGA CA_KI_H2CGGATCCCAAGCTTC (SEQ ID No 90)

Transformation.

Plasmids were transformed in CaCl₂ competent cells using the simplifiedprocedure of Hanahan (16) or via electroporation as described above.

Calculation Methods Introduction

Different experiments with different strains were performed. In total 8different conditions were tested. There was variation in the geneticbackground (WT, iclR knock-out, arcA knock-out, and combined iclR-arcAknock-out) and the mode of fermentation (batch, and chemostat). Eachexperiment was repeated twice.

When running the samples through the BioTrove apparatus, a qPCR curve(fluorescences in function of cycle number) and a melt curve(fluorescences in function of the temperature) is obtained for eachsample. Those data were exported from the BioTrove software and furtheranalysed in R. The analysis was divided in two steps: first the qPCRcurves were fitted and Ct values were calculated and in the second stepthe Ct values were converted to expression data.

Calculating the qPCR Curves

The raw qPCR curve data were extracted from the BioTrove software andimported in R (1). The curves were fitted to a 5 parameter sigmoidalmodel, with the R package qPCR (25, 34). The maximum of the secondderivative of those curves was used as Ct value. No normalisation wasapplied to the data prior to the curve fitting. However, outliers wereremoved. The detection of the outliers was done using the followingprocedure:

-   -   Fit the model to the data.    -   Calculate the residuals (defined as the measured fluorescences        minus the model-calculated ones).    -   Assuming the residuals are normally distributed, calculate the        mean and standard deviation of the residuals.    -   Using this mean and standard deviation, the 95% interval is        calculated.    -   All data-points for which the residuals fall out of this 95%        interval are considered as outliers.    -   The curve is refitted without the outliers.    -   This is repeated until no outliers are detected anymore. Using        this procedure, the data do not have to be normalised prior to        fitting, neither must the first data-points be removed.

Many curves have to be fitted (1800 genes for one experiment).Therefore, it is undoable to manually check each curve and automatedmethods have to be applied to reject bad curves. For this differentparameters are extracted from the curves: the cycle number value atwhich the maximum of the first derivative occurs (D1), the cycle numbervalue at which the maximum of the second derivative occurs (D2), theminimal fluorescence (Fmin), and the maximal fluorescence (Fmax).Combining the values of those parameters, the validity of the curve andthe extent of expression is assessed. How this is done is explained inthe next section.

Filtering the Data

For some gene-experiment combinations, no amplification is detected.This can be due to a variety of reasons:

-   -   Expression is too low and 32 cycles (the number of cycles for        all BioTrove arrays was set to 32) is not enough to detect the        expression. In this case, the real Ct cannot be determined and        is somewhere between 32 and infinity.    -   No expression. In this case, the real Ct is infinite.    -   Technical failures: primers not suitable, wrong loading (it is        very difficult to uniformly load the BioTrove arrays, especially        the holes at the sides of the array are frequently empty), etc.        In this case the real Ct can vary between 0 and infinity.

Some genes are genuinely not expressed and setting their Ct value tosomething else than infinity is not correct. For genes that areexpressed, but for which the expression value, due to technical failuresor limitations, are not known, setting the Ct value to infinity is notcorrect. Furthermore, using arbitrary values that are outside the rangeof expression complicates the calculation routines and visualisationroutines. Therefore it was opted to remove the gene-experimentcombinations for which no correct expression data was detected.

An obvious case of gene-experiment pairs for which no expression isdetected, are those for which no curve could be fitted to the qPCR data.Less obvious cases are detailed below.

Typically for expressed genes, is that the fluorescence values cover acertain range. Data points for which this range was not high enough,were discarded, as they pointed to very poorly fitted curves andgenerally bad data. The minimal fluorescence range was set to 400 (thusFmax−Fmin>400).

In a good amplification curve, the first (D1) and second (D2) derivativeare quite close to each other (see the documentation of the SOD functionin the qpcR package(25)). Therefore, all data-points for which thedifference between D1 and D2 is larger than an arbitrary value (7 wasused) were discarded.

For each primer-pair, a qPCR experiment was performed without addingDNA. Only water was added. Normally no expression should be observed inthose samples. However, amplification is detected in water for someprimer-pairs. Genes for which the Ct value (as mentioned before, D2 wasused) is more than the Ct value of water minus 5, are discarded, as itcannot be excluded that the fluorescence comes from the amplification ofthe primers and not the added DNA.

Normalising and Calculating the Contrasts

Prior to calculating the expression differences, the Ct values have tobe normalised. As so many genes were measured (1800), quantilenormalisation could be used (33). The 1800 genes measured, were dividedover 3 types of arrays, each containing 600 genes. Quantilenormalisation was done for each type of array separately. A table wasconstructed where the rows represent the different genes and the columnsthe different experiments (T1, see Equations 1). Each column was sortedindependently (T2) and the original position of the elements was saved.The values in this new table were replaced with the mean value over thedifferent rows (T3). And finally this table was transformed so that thepositions of the values corresponded again to the original positions(T4).

$T_{1} = \begin{bmatrix}2 & 4 \\6 & 8 \\4 & 12\end{bmatrix}$ $T_{2} = \begin{bmatrix}2 & 4 \\6 & 8 \\4 & 12\end{bmatrix}$ $T_{3} = \begin{bmatrix}3 & 3 \\6 & 6 \\9 & 9\end{bmatrix}$ $T_{4} = \begin{bmatrix}3 & 3 \\6 & 6 \\9 & 9\end{bmatrix}$

Equations 1: Example of Quantile Normalisation

Differential expressions were calculated with the normalised data. Thiswas done with the R package limma, which uses a Bayesian approach tocalculate the statistical relevances of the differences (31, 32). Limmawas adapted to be able to cope with missing data: the original limmapackage discards all expression values from a gene over the differentexperiments, when one value in one experiment is not available. Thishampers the analysis when one has many different conditions, as for eachgene for which one of the experimental conditions produces no expressionvalues, a different contrast matrix has to be generated omitting thatexperimental condition. Therefore the function for fitting the contrastswas adapted to drop data-points with missing data.

Differential expressions were calculated between Ct values and the meanCt value for a certain gene. Thus, the higher the value, the lower theexpression. For each gene, plots were generated showing thosedifferences. However, in those plots, the Ct values were inversed, sothat the higher the value, the higher the expression.

Example 1: Effect of arcA and iclR Gene Deletions on the Gene Expressionof the Colanic Acid Biosynthesis

FIGS. 1 and 2 show the expression pattern of genes involved in colanicacid biosynthesis (35). Single arcA or iclR knock out mutations did notaffect the expression of the operon in comparison of the wild typestrain in batch and chemostat conditions. The double mutant strain,ΔarcAΔiclR, however upregulates the genes of the colanic acid operon 6to 8 times in comparison to the wild type and the single mutant strainsin both chemostat and batch conditions. Both regulators have thus asurprisingly cooperative effect on the expression of this operon whichis independent from the culturing condition that is applied. Looking atthe regulatory network of this operon, no direct link could be foundbetween both ArcA and IclR and the transcription factor that controlsthe operon, RcsA (FIG. 5). Only ArcA is connected with RcsA via 3 othertranscription factors, which are all upregulated as well. However theΔarcA single gene deletion mutant strain did not affect thetranscription of the operon.

Example 2: Effect of arcA and iclR Gene Deletions on the Gene Expressionof the GDP-Fucose Biosynthesis Genes

FIGS. 4 and 6 show the relationship of the colanic acid operon withGDP-fucose biosynthesis. In FIG. 6 the upregulation of GDP-fucosebiosynthesis specific genes is shown. These mutations thus enhance thebiosynthesis of GDP-fucose, which is a precursor for fucosylatedoligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis Xoligosaccharide or fucosylated proteins. These sugars and proteins, asalready indicated above, have applications in therapeutics asnutraceutical, as components in human mother milk in which they haveanti-inflammatory and prebiotic effects (5, 8, 27).

Example 3: Enhancement of GDP-Fucose and Fucosylated OligosaccharideBiosynthesis

The mutations ΔarcAΔiclR applied in combination with other mutationsenhance the production of fucosylated compounds. A first, ‘other’genetic modification that enhances said production is the deletion ofwcaJ from the colanic operon, stopping the initiation of the colanicacid biosynthesis and thus the accumulation of GDP-fucose. Further, afucosyltransferase has to be introduced to link fucose with differentacceptor molecules such as lactose. The metabolism is then engineeredfurther to accumulate the precursor of the GDP-fucose biosyntheticpathway. These modifications are shown in FIG. 7. Additional to wcaJ,the colanic acid operon genes that do not code for GDP-fucosebiosynthesis reactions are knocked out, such as gmm, wcaA, wcaB, wcaC,wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM. For the production offucosyllactose, lacZ coding for β-galactosidase, is knocked out to avoidlactose degradation and the expression of lacY, coding for a lactosepermease, is enhanced by means of a strong constitutive promoter.

Example 4: Enhancement of GDP-Fucose and Fucosylated OligosaccharideProduction Via a Split Metabolism with Sucrose as a Substrate

To accumulate the GDP-fucose precursor fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced. Because fructose-6-phosphate is easily degraded in theglycolysis, the glycolysis is interrupted in order to steer allfructose-6-phosphate in the direction of GDP-fucose. The genes pgi, pfkAand pfkB are thus knocked out, coding for glucose-6-phosphate isomeraseand phosphofructokinase A and B. Finally a fucosyltransferase isintroduced to link fucose to an acceptor molecule.

The growth rate of the wild type strain is somewhat affected when grownon sucrose after introduction of a sucrose phosphorylase (BaSP) (plasmidwith sequence SEQ ID No 2) (Table 2), however the introduction of pgimutations and pfkA and pfkB double mutations led to significantreduction of growth rate, the latter was extremely low (0.02 h⁻¹). Thecombination of all mutations (Δpgi and ΔpfkA and ΔpfkB) led to thelowest growth rate, however, the growth rate on both sucrose and glucosewas surprisingly similar to that of the pgi single mutant.

TABLE 2 specific growth rates of the glycolysis knock out strains on aminimal medium with glucose and sucrose Growth rate on sucrose (h⁻¹)Growth rate on (strains transformed with Strain glucose (h⁻¹) plasmidcontaining BaSP) Wild type 0.64 0.41 Δpgi 0.18 0.23 ΔpfkAΔpfkB 0.02 n.d.ΔpgiΔpfkAΔpfkB 0.23 0.24

SEQ ID No 2: Plasmid sequence with sucrose phosphorylase BaSPAATTCGGAGGAAACAAAGATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGAAAAACAAGGTGCAGCTCATCACTTACGCCGACCGCCTTGGCGACGGCACCATCAAGTCGATGACCGACATTCTGCGCACCCGCTTCGACGGCGTGTACGACGGCGTTCACATCCTGCCGTTCTTCACCCCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAGGTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAGACCCACAACATCATGGTCGACGCCATCGTCAACCACATGAGTTGGGAATCCAAGCAGTTCCAGGACGTGCTGGCCAAGGGCGAGGAGTCCGAATACTATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCGAACGGCGCCACCGAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTCACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTCACCCCGCAGCAGGTGGACATCGACACCGATTCCGACAAGGGTTGGGAATACCTCATGTCGATTTTCGACCAGATGGCCGCCTCTCACGTCAGCTACATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAAGCCGGCACCAGCTGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAGGAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTACTACAAGAAGCAGGTCGAAATCGCATCCAAGGTGGACCGCGTCTACGACTTCGCCCTGCCTCCGCTGCTGCTGCACGCGCTGAGCACCGGCCACGTCGAGCCCGTCGCCCACTGGACCGACATACGCCCGAACAACGCCGTCACCGTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGACCAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGACAACCTCGTCAACACCATCCACGCCAACACCCACGGCGAATCCCAGGCAGCCACTGGCGCCGCCGCATCCAATCTCGACCTCTACCAGGTCAACAGCACCTACTATTCGGCGCTCGGGTGCAACGACCAGCACTACATCGCCGCCCGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTCGGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGCGTAAGACGAATAACGGCCGCGACATCAATCGCCATTACTACTCCACCGCGGAAATCGACGAGAACCTCAAGCGTCCGGTCGTCAAGGCCCTGAACGCGCTCGCCAAGTTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTCTCGTACACCACCGATGACGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCCAGGCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACGCTACGCCGGTCGCCATGTTGGAATGGGAGGATTCCGCGGGAGACCACCGTTCGGATGATCTGATCGCCAATCCGCCTGTCGTCGCCTGACTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATCCCGGGCGGCCGCTTCATTTATAAATTTCTTGACATTTTGGAATAGATGTGATATAATGTGTACATATCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATC CGTCGACCTCG

The flux redirections and mutations for GDP-fucose and fucosylatedoligosaccharide biosynthesis in a split metabolism are shown in FIG. 8,both for a strain expressing a heterologous invertase and sucrosephosphorylase. Additional to wcaJ, the colanic acid operon genes that donot code for GDP-fucose biosynthesis reactions are knocked out, such asgmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM.For the production of fucosyllactose, lacZ, coding for β-galactosidase,is knocked out to avoid lactose degradation and the expression of lacY,coding for a lactose permease, is enhanced by means of a strongconstitutive promoter.

Example 5: Enhancement of GDP-Fucose and Fucosylated OligosaccharideProduction Via a Split Metabolism with Glucose as Substrate

When the genes pgi, pfkA, and pfkB are knocked out, carbon, taken up asglucose can only be metabolised via the pentose phosphate pathway. Dueto the biochemical properties of this pathway, fructose-6-phosphate isformed (FIGS. 9 and 10). To form biomass glyceraldehyde-3-phosphate hasto be formed, which is formed by the transketolase reactions coded bytktA and tktB in E. coli. This Glyceraldehyde-3-phosphate is formedtogether with fructose-6-phosphate from xylulose-5-phosphate anderythrose-5-phosphate. The latter is in turn formed together withfructose-6-phosphate from glyceraldehyde-3-phosphate andsedoheptulose-7-phosphate via transaldolase reactions coded by talA andtalB. To balance all of these reactions together the flux has to bedistributed between xylulose-5-phosphate and ribose-5-phosphate, as suchthat from 1 mole glucose, ⅔ mole of xylulose-5-phosphate and ⅓ moleribose-5-phosphate is formed. To drive these equilibrium reactions,fructose-6-phosphate is pulled out of the pentose phosphate pathway bythe GDP-fucose and fucosylated oligosaccharide biosynthesis pathway.Additional to wcaJ, the colanic acid operon genes that do not code forGDP-fucose biosynthesis reactions are knocked out, such as gmm, wcaA,wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM. For theproduction of fucosyllactose, lacZ coding for β-galactosidase, isknocked out to avoid lactose degradation and the expression of lacY,coding for a lactose permease, is enhanced by means of a strongconstitutive promoter.

Example 6: Fermentative 2-Fucosyllactose Production with aFucosyltransferase Originating from Helicobacter pylori

The mutant strain in which the genes lacZ, glgC, agp, pfkA, pfkB, pgi,arcA, iclR, wcaJ are knocked out and lacY was expressed via constitutiveexpression to ensure expression under all culturing conditions, wastransformed further with a fucosyltransferase originating fromHelicobacter pylori and a sucrose phosphorylase originating fromBifidobacterium adolescentis, which were also constitutively expressed.The constitutive promoters originate from the promoter library describedby De Mey et al. 2007. This strain was cultured in a medium as describedin the materials and methods, however with 30 g/l of sucrose and 50 g/lof lactose. This resulted in the formation of 2-fucosyllactose as shownin FIGS. 13 and 14.

Example 7: Fermentative Fucosyllactose Production with aFucosyltransferase Originating from Dictyostellium discoideum

The mutant strain in which the genes lacZ, glgC, agp, pfkA, pfkB, pgi,arcA, iclR, wcaJ are knocked out and lacY was expressed via constitutiveexpression to ensure expression under all culturing conditions, wastransformed further with a fucosyltransferase originating fromDictyostellium discoideum and a sucrose phosphorylase originating fromBifidobacterium adolescentis, which were also expressed constitutively.The constitutive promoters originate from the promoter library describedby De Mey et al. 2007. This strain was cultured in a medium as describedin the materials and methods, however with 30 g/l of sucrose and 50 g/lof lactose. This resulted in the formation of 2-fucosyllactose as shownin FIGS. 13 and 14.

Example 8: Enhancement of GDP-Mannose and Mannosylated OligosaccharideProduction Via a Split Metabolism with Sucrose as Substrate

To accumulate the GDP-mannose precursors fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced. Because fructose-6-phosphate is easily degraded in theglycolysis, the glycolysis is interrupted in order to steer allfructose-6-phosphate in the direction of GDP-fucose. The genes pgi, pfkAand pfkB are thus knocked out, coding for glucose-6-phosphate isomeraseand phosphofructokinase A and B. Finally a mannosyltransferase isintroduced to link mannose to an acceptor molecule. To avoid GDP-mannosedegradation the genes gmm and gmd have to be knocked out in the colanicacid operon. In addition, the genes that do not code for GDP-mannosebiosynthesis reactions are knocked out, such as wcaA, wcaB, wcaC, wcaD,wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL and/or, wcaM.

Example 9: Upregulation of Acid Resistance Related Genes

Similar to the colanic acid operon upregulation, acid resistance relatedgenes are also upregulated in a ΔarcAΔiclR double mutant strain incomparison to the wild type strain and the single mutant strains. Thesegenes make a strain more resistant to low pH, which is beneficial forthe production of acids (4) or the production of glucosamine (12) whichis not stable at neutral and high pH. FIG. 12 presents the geneexpression pattern of these acid resistance related genes and indicatesup to 8 fold expression increase in the double mutant strain.

Example 10: Fed Batch Production of 2-Fucosyllactose

A mutant strain was constructed via the genetic engineeringmethodologies described above with the following genotype:

ΔlacZYA::P22-lacYΔglgCΔagpΔpgiΔpfkA-P22-baSPΔpfkBΔarcAΔiclR::slΔwcaJΔlonΔadhE-P14-frk+pCXP14-FT_H.pylori (a vector with sequence SEQ ID No 1). The promoter P22 and P14originate from the promoter library constructed by De Mey et al (11) andwas cloned similar to the methodology described by Aerts et al (2).“::sl” marks a scarless gene deletion, thus without a FRT site thatremains in the chromosome.

This strain was cultured in a bioreactor as described above in materialsand methods, in the mineral medium with 30 g/l of sucrose and 50 g/l oflactose. After the batch phase the bioreactor was fed with 500 g/l ofsucrose, 50 g/l lactose and 1 g/l of magnesium sulphate heptahydrate.This led to the accumulation of 27.5 g/l of fucosyllactose in thesupernatant.

SEQ ID No 1: pCXP14-FT_H. pyloriCGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATCCCGGGCGGCCGCCTTCATTCTATAAGTTTCTTGACATCTTGGCCGGCATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCGAATTCGGAGGAAACAAAGATGGCCTTTAAAGTTGTTCAGATTTGTGGTGGTCTGGGCAATCAGATGTTTCAGTATGCATTTGCAAAAAGCCTGCAGAAACATAGCAATACACCGGTTCTGCTGGATATTACCAGCTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTTTCCGATTGATCTGCCGTATGCAAGCGAAAAAGAAATTGCAATTGCCAAAATGCAGCATCTGCCGAAACTGGTTCGTAATGTTCTGAAATGCATGGGTTTTGATCGTGTGAGCCAAGAAATCGTGTTTGAATATGAACCGAAACTGCTGAAAACCAGCCGTCTGACCTATTTTTATGGCTATTTTCAGGATCCGCGTTATTTTGATGCAATTAGTCCGCTGATCAAACAGACCTTTACCCTGCCTCCGCCTCCGGAAAATGGTAATAACAAAAAAAAAGAAGAAGAGTATCATCGTAAACTGGCACTGATTCTGGCAGCAAAAAATAGCGTGTTTGTGCATATTCGTCGCGGTGATTATGTTGGTATTGGTTGTCAGCTGGGCATCGATTATCAGAAAAAAGCACTGGAATACATGGCAAAACGTGTTCCGAATATGGAACTGTTTGTGTTTTGCGAGGACCTGGAATTTACCCAGAATCTGGATCTGGGCTATCCGTTTATGGATATGACCACCCGTGATAAAGAGGAAGAGGCATATTGGGATATGCTGCTGATGCAGAGCTGTAAACATGGTATTATTGCCAACAGCACCTATAGTTGGTGGGCAGCATATCTGATTAATAACCCGGAAAAAATCATTATTGGTCCGAAACATTGGCTGTTTGGCCATGAAAACATCCTGTGTAAAGAATGGGTGAAAATCGAAAGCCACTTTGAAGTGAAAAGCCAGAAATATAATGCCTAATAAGAGCTCCCAA 

Example 11: Fed Batch Production of 2-Fucosyllactose with a HybridColanic Acid Promoter

A hybrid colanic acid promoter was constructed based on the genomeinformation and the sequences from the promoter library described by DeMey et al (11).

ΔlacZYA::P22-lacYΔglgCΔagpΔpgiΔpfkA::P22-BaSPΔpfkB ΔarcAΔiclR:sl ΔwcaJΔlon ΔadhE-P14-frk ΔldhA::P14-FT_H. pylori ΔpromCA:P14

This strain was cultured in a bioreactor as described above in materialsand methods, in the mineral medium with 30 g/l of sucrose and 20 g/l oflactose. After the batch phase the bioreactor was fed with 500 g/l ofsucrose, 20 g/l lactose and 1 g/l of magnesium sulphate heptahydrate.This led to the accumulation of 26 g/l of fucosyllactose in thesupernatant with nearly stoichiometric conversion of lactose. Increasingthe lactose feed concentrations leads further to increased finalfucosyllactose titers and stoichiometric lactose conversion.

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1. A mutated and/or transformed organism in which regulators ArcA andIclR in combination with genes encoding for enzymes phosphoglucoseisomerase and phosphofructokinase are knocked out or are rendered lessfunctional.
 2. A mutated and/or transformed organism according to claim1, wherein the enzyme phosphoglucose isomerase is encoded by gene pgiand wherein enzyme phosphofructokinase is encoded by gene(s) pfkA and/orpfkB.
 3. A mutated and/or transformed organism according to claim 1,wherein said organism is further transformed with a gene encoding for asucrose phosphorylase or invertase.
 4. A mutated and/or transformedorganism according to claim 1, wherein the activity of a gene encodingfor a lactose permease is increased.
 5. A mutated and/or transformedorganism according to claim 1, wherein at least one of the followinggenes is knocked out or is rendered less functional: a gene encoding fora beta-galactosidase, a gene encoding for a glucose-1 phosphateadenylyltransferase, a gene encoding for a glucose-1-phosphatase, a geneencoding for phosphogluconate dehydratase, a gene encoding for2-keto-3-deoxygluconate-6-phosphate aldolase, a gene encoding for aglucose-1-phosphate uridyltransferase, a gene encoding for anUDP-glucose-4-epimerase, a gene encoding for anUDP-glucose:galactose-1-phosphate uridyltransferase, a gene encoding foran UDP galactopyranose mutase, a gene encoding for an UDP galactose:(glucosyl)lipopolysaccharide-1,6-galactosyltransferase, a gene encodingfor an UDP-galactosyltransferase, a gene encoding for anUDP-glucosyltransferase, a gene encoding for an UDP-glucuronatetransferase, a gene encoding for an UDP-glucose lipid carriertransferase, a gene encoding for a GDP-mannose hydrolase, a geneencoding for an UDP-sugar hydrolase, a gene encoding for amannose-6-phosphate isomerase, a gene encoding for anUDP-N-acetylglucosamine enoylpyruvoyl transferase, a gene encoding foran UDP-Nacetylglucosamine acetyltransferase, a gene encoding for anUDP-Nacetylglucosamine-2-epimerase, a gene encoding for anundecaprenyl-phosphate alfa-N-acetylglucosaminyl transferase, a geneencoding for a glucose-6-phosphate-1-dehydrogenase, and/or, a geneencoding for a L-glutamine:D-fructose-6-phosphate aminotransferase, agene encoding for a mannose-6-phosphate isomerase, a gene encoding for asorbitol-6-phosphate dehydrogenase, a gene encoding for amannitol-1-phosphate 5-dehydrogenase, a gene encoding for aallulose-6-phosphate 3-epimerase, a gene encoding for an invertase, agene encoding for a maltase, a gene encoding for a trehalase, a geneencoding for a sugar transporting phosphotransferase, a gene encodingfor a protease, or a gene encoding for a hexokinase.